The parathyroid hormone receptor (PTHR) is a G protein-coupled receptor that initiates intracellular signaling in response to parathyroid hormone (PTH) and parathyroid hormone related protein (PTHrP). PTH-induced signaling in bone and kidney promotes physiological responses related to systemic control of mineral and skeletal homeostasis, whereas paracrine signaling initiated by PTHrP in developing cartilage regulates the timing of cartilage differentiation. Moreover, when appropriately administered, PTH acts through the PTHR to produce anabolic effects on the skeleton and may have therapeutic value in patients with osteopenic disorders. It is therefore essential to understand the mechanisms by which the PTHR initiates signal transduction and how these signaling processes are regulated. Regulation of the function of the PTHR is known to involve both desensitization of receptor signaling and down-regulation of receptor number, but the mechanisms underlying these processes are unclear. Three specific aims are proposed. Aim 1 is to examine the role of PTHR phosphorylation and of arrestin proteins in the regulation of PTHR signaling. Mutated PTHRs lacking the sites of PTH-stimulated phosphorylation have been identified, and these will be used explore the role of phosphorylation in PTHR desensitization. Studies will be carried out to determine whether arrestin proteins interact with PTHRs, and whether disruption of arrestin function blocks PTHR desensitization or endocytosis. Aim 2 is to evaluate the role of PTHR phosphorylation in post-endocytic receptor trafficking. Confocal microscopy will be used to track endocytosis and recycling of wt and phosphorylation-deficient PTHRs labeled with the green fluorescent protein. The role of PTHR phosphorylation in PTH-induced down-regulation of the receptor will also be investigated. Aim 3 is to elucidate the mechanisms and significance of rapid agonist-induced endocytosis of the PTHR. Positive endocytic sequences that mediate rapid internalization of the PTHR via clathrin-coated pits will be identified. The yeast two-hybrid system will be used to identify cellular proteins that bind to these motifs and mediate receptor endocytosis. Mutated, endocytosis-deficient PTHRs will be used to assess a positive role of endocytosis in PTHR resensitization and activation of intracellular signaling pathways. Successful completion of these studies will provide new mechanistic insights into the regulatory mechanisms by which cells control the signaling properties of the PTHR.